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International Journal of Advanced Research in Biological Sciences Int. J. Adv. Res. Biol. Sci. (2017). 4(12): 292-299 International Journal of Advanced Research in Biological Sciences ISSN: 2348-8069 www.ijarbs.com DOI: 10.22192/ijarbs Coden: IJARQG(USA) Volume 4, Issue 12 - 2017 Research Article DOI: http://dx.doi.org/10.22192/ijarbs.2017.04.12.032 Taxonomic characterization of the chitinolytic actinomycete Cellulomonas chitinilytica strain HwAC11 Gamal M. El-Sherbiny1, Osama M. Darwesh2*, Ahmad S. El-Hawary1 1Botany and Microbiology Department, Faculty of Science (Boys); Al-Azhar University, Nasr City, Cairo, Egypt. 2Agricultural Microbiology Department, National Research Centre, Cairo, Egypt. *Corresponding author: E-mail: [email protected] Mobile: +201155265558, Fax: +20237601036 Abstract Chitinases apply in several useful fields such as agriculture, food industries and environmental applications. Because it helps degradation of fungal cell walls containing chitin and thus accelerates protoplast formation. The alkaliphilic action-bacterial strain HwAC11 was isolated from compost after examine 99 different samples. This isolate exhibited good growth on medium containing chitin as sole carbon source. Macro- and micro–morphological characteristics, enzyme activities, physiological and biochemical properties of the isolate were investigated. It was concluded that the strain HwAC11 is a member of the genus Cellulomonas. The results were compared with the taxonomic characteristics of Cellulomonas members and it was found to be similar to those of Cellulomonas chitinilytica. The phylogenetic analysis based on 16s ribosomal RNA gene sequence confirmed the phenotypic results and the sequences were deposited in gene bank under Cellulomonas chitinilytica strain HwAC11with accession number MH050787. The strain HwAC11 displayed intensive chitinase activity under alkaline conditions. It leads to apply this strain in agriculture field, especially as biological control agent for pathogenic fungi and harmful nematodes. Keywords: Alkaliphilic action-bacteria, Taxonomy, 16S rDNA, Cellulomonas chitinilytica, Chitinase. Introduction Enzymes hydrolyze Polysaccharides as chitinases, Chitinases are glycoside hydrolases that degrade chitin cellulases and pectinases are widespread in nature. by hydrolyzing the β-1,4-glycosidic linkages. They can be found in all living forms, including Chitinase enzyme activity has ability to degrade the animals, plants, algae, fungi and bacteria (Yoon et al., cell wall of pathogens containing chitin such as insect 2008). To produce polysaccharides, micro-organisms exoskeleton, nematodes and fungi cell wall. Therefore, are usually the most convenient sources and can be controlling of insects and pathogenic fungi by obtained from various natural environments Chitin, the chitinases are powerful biological tools. In addition, insoluble linear β-1,4-linked N-acetylglucosamine, is the pathogens fail to develop resistance as enzymes the most abundant polysaccharide in nature after are not persistent against them. Many studies have cellulose, with production of 100 billion tons annually reported that chitinolyticbacteria inhibit the mycelial (Yu et al., 1993). 292 Int. J. Adv. Res. Biol. Sci. (2017). 4(12): 292-299 extension of several pathogenic fungi (Chernin et al., of the extract and 1 ml of 10% (w/v) suspension of 1995; Prasanna et al., 2013). Consequently, bacterial colloidal chitin, in 0.2 M potassium phosphate buffer chitinases have a critical role in the breakdown of (pH 8.5), and incubated at 30 ºC for 1 h. The reaction chitin in cell wall of fungi, and chitinase producing was stopped by adding 1 ml of 1% NaOH, followed by bacteria could be widely applied as ecofriendly agents boiling for 5 min. Tubes were then centrifuged at 7000 against agricultural phytopathogens (Rahman et al., rpm and the reducing sugars produced were 2016; Kamensky et al., 2003; Kurzeet al., determined in the supernatants. One milliliter of 2001).Genus Cellulomonas is a member of the family supernatant and 1 ml of 1% DNS (dissolved in 30% cellulomonadaceae in the phylum action-bacteria. sodium potassium tartrate in 2 M NaOH) were mixed Species ofgenus Cellulomonas have typical and incubated for 5 min in a boiling water bath. characteristics in that are Gram positive rods with Afterward, their absorbance at 535 nm was recorded. cellulolytic activity (Stackebrandt et al., 2014). Also, Readings were interpolated in a standard curve few members are known to have chitinolytic activity prepared with a series of dilutions (0– 10 μmol/ml) of as Cellulomonas chitinilytica. For that, this study was N-acetyl-D-glucosamine (NAG) and DNS. The concerned with isolation of alkali chitinolytic action- chitinase activity was defined as the amount of bacteria from Egyptian environment and taxonomic enzyme required to produce one μmol of NAG in 1 h characterization of the most potent isolate depending (Rojas Avelizapa et al., 1999). The action-bacterial on its enzyme activity. isolate showing the highest chitinase activity was selected to investigate its characterizations. Materials and Methods Morphological characteristics Isolation and culture conditions of chitinolytic bacteria The micro-morphological and cultural properties of the most active isolate were investigated according to A number of 99 samples of soil, composts and the international Streptomyces project (ISP) after 24, sediments were collected during 2016 from different 48, 72 h and one week of cultivation. The observations environment sites in Egypt (Baharia Oasis, and records of colors were described as in the ISP Alexandria, Alfayoum, and Wadi Alnatroun).The methods (Shirling & Gottlieb, 1966). The ISCC-NBS samples were aseptically collected in clean plastic color name charts illustrated with centroid color bags at depth 15-20 cm. The collected samples were (Kenneth and Deane, 1955) was used for this sieved to remove various contaminant materials. Then purpose. The morphology of the spore chains and it was air-dried and mixed with CaCO3 (1g/ 100g soil) sporophores was studied by direct light microscopy for 24 hours at 28° C before plating to increase the observation. The morphology of the spores was numbers of action-bacteria (Tsao et al., 1960). examined by scanning electron microscopy (Tresner Isolation of chitinolytic action-bacteria was performed et al., 1961). by direct inoculation of 1 g of each sample to basal agar medium (w/v, 0.5% (NH4)2SO4, 0.085% KH2PO4, Physiological and biochemical properties 0.015% K2HPO4, 0.05% MgSO4, 0.01% NaCl, 0.01% CaCl2, pH 8.5, 1.5% agar) containing 0.2% (w/v) Growth of action-bacterial isolate HwAC11 was colloidal chitin and incubated at 30 °C for 7 days. assessed in nutrient agar (NA) (Shirling & Gottlieb, Selected colonies (rough, chalky) of action-bacteria 1966) (g/l) ( Beef extract, 3.0; peptone, 5.0; sodium were transferred onto respective agar plates and chloride, 5.0; Agar, 20.0; Distilled water 1000 ml) incubated for 7 days. Plates containing pure cultures andstarch-nitrate agar medium (SNA) (Tadashi, 1975) were stored until further examinations. (g/l) (Soluble starch, 20.0; NaNO3, 2.0; K2HPO4, 1.0; KCl, 0.5; MgSO4.7H2O, 0.5; CaCO3.2H2O, 2.0; Agar, Chitinase activity assay 20.0; Distilled water up to1000 ml) at 10, 20, 30, 40, 50 °C and pH 4.0–10.0 (at 1.0 pH unit intervals) Isolated chitinolytic action-bacteria were grown on adjusted by using HCl and NaOH (Gomori, 1955). basal medium containing 1% colloidal chitin for 7 The tolerance to NaCl was tested on starch-nitrate agar days (30 °C, 150 rpm). Cells were separated by medium containing different concentrations of NaCl centrifugation (4000 rpm, 10 min, 4 °C) and the from 0 to 3.5 % (w/v). supernatant was assayed. The chitinase activity assay was conducted in a reaction containing one milliliter 293 Int. J. Adv. Res. Biol. Sci. (2017). 4(12): 292-299 Utilization of different substrates for growth In-silico DNA sequence analyses were carried out by Sanger sequencing technology on applied bio-systems The utilization of various carbon and nitrogen sources automated DNA sequencer, model ABI 3730XL DNA was tested on basal mineral salts agar (ISP-9) Analyzer (Applied Bio-systems, USA; service (Shirling and Gottlieb, 1966) supplemented with provided by Macrogen Inc., South Korea). The different organic substrates (1 %, w/v). sequence analyses and alignments were performed by NCBI-BLAST programs of the National Center for Enzyme activity Biotechnology Information (Altschul et al., 1990; Stultz et al., 1993; Hobohm and Sander, 1995 and Hydrolytic enzymes activity was investigated using Altschul et al., 1997). Multiple sequence alignment basal mineral salts agar medium (ISP-9)supplemented and molecular phylogeny were performed using with 1% (w/v) starch (Kurup, et al., 1975), casein BioEdit software (Hall, 1999). The phylogenetic tree (Gordon & Smith, 1955), carboxy-methyl cellulose was displayed using the TREEVIEW program (Page, (CMC) (Teather & Wood, 1982), pectin (Venkata 1996). et al., 2013) and tributyrin (Heravi et al., 2008) for amylase, protease, cellulase, pectinase and lipase Results enzymes assay, respectively. Other physiological and biochemical studies were conducted according to Isolation of chitinolytic action-bacteria El-Sherbiny et al. (2017) A total of 99 samples were collected, and the variety Phylogenetic analysis of samples types and ecology were taken in account to increase the diversity of the isolated microorganisms. Genomic DNA extraction was performed by After inoculation and incubation, 8 different isolates lysozyme-sodium dodecyl
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